Sound pressure produces force across the mammalian cochlear partition, ultimately creating a vibratory traveling wave that propagates longitudinally up the cochlear duct. The key feature distinguishing this process from the non-mammalian cochlea is amplification, whereby forces produced by thousands of outer hair cells (OHCs) sharpen and amplify the traveling wave. Our overarching objective is to understand how the complex biomechanics of the 3D multi-cellular and acellular arrangement that forms the organ of Corti work together to create cochlear amplification. Specifically, we will determine how this process, which stems from the broadly- tuned basilar membrane, creates sharp frequency tuning and high sensitivity. This question is significant on a basic science level because these biophysical processes underlie the ability to hear sounds just above the Brownian motion of molecules in air with an exquisite frequency resolution. This question remains unsolved and is clinically important because hearing loss is typically due to loss of cochlear amplification. Our central hypothesis is that the mechanical properties of the organ of Corti provide additional filtering beyond that provided by the passive mechanics of the basilar membrane and surrounding fluid, and that this modulates OHC force production to give rise to the observed sensitivity and sharp frequency tuning. To test the hypothesis, we have developed an innovative technology, termed Volumetric Optical Coherence Tomography Vibrometry (VOCTV). Besides permitting traditional basilar membrane motion measurements in vivo, VOCTV also permits the measurement of sound-induced vibrations throughout the organ of Corti. We propose to use VOCTV to study the interactions between components of the organ of Corti and assess how they relate to cochlear amplification within the apical turn of the mouse cochlea. In Aim 1, we propose to measure transverse and radial vibratory motions within the apical turn of the wild- type mouse cochlea in vivo for the first time. We will use 3D localization to compare the responses of different organ of Corti structures, assessing both the frequency response and the gain of cochlear amplification. In Aim 2, we propose to measure vibratory motions within the apical turn of several transgenic mouse strains that have molecular changes designed to selectively alter of organ of Corti mechanics. Through this approach, we will probe the mechanical contributions of prestin-based electromotility, stereociliary bundle mechanics, tectorial membrane traveling waves, and hair cell/supporting cell patterning. Together, these data will be interpreted so as to test our hypothesis. If our hypothesis is true, sharply-tuned differential motion within the organ of Corti is necessary to generate the sensitivity and sharp tuning of the mammalian cochlea.